Production and partial purification of α-amylase from Pseudomonas sp. 2 under solid-state fermentation

Abstract

Purpose: The purpose of the present study was to isolate Pseudomonas sp. 2 from rhizosphere and screen for best amylase producer and to check for its suitability for industrial applications. Methods: Four strains of Pseudomonas were isolated from rhizosphere soil, by serial dilution method, of which Pseudomonas sp. 2 gave best results to α-amylase production. This strain was used to determine the enzyme production by SSF on wheat bran. Results and conclusion: The optimal enzyme production was obtained at 37 ºC and pH 7.5. Vitamins B 12 (50 mg) followed by Pyridoxine (50 mg) were found to be enhancing the enzyme production. Amino acids cysteine (30 mg), tyrosine (40 mg) and alanine (30 mg) were stimulating the enzyme production. The enzyme was partially purified by ammonium sulphate precipitation, and was further purified by ion exchange DEAE-cellulose column Chromatography. The purity of the enzyme was 45.2 fold greater than the crude enzyme. The molecular weight of the enzyme was found to be ~62 kDa by SDS-PAGE. This is the first report of any Pseudomonas sp. from rhizosphere being used for amylase production. The purified enzyme was not thermostable but it was acid tolerant making it suitable for industrial application. Key words: Pseudomonas sp. 2, α-amylase, SSF, Vitamins, Amino acids, Partial purification. Conflict of interest:The authors declare no conflicts of interest of any kind. ÖZETAmaç: Çalışamanın amacı Rizofer'den Pseudomonas sp. 2 bakterisini izole etmek, bundan en iyi amilaz üretimini bulmak ve bunun endüstriyel aplikasyonlara uygunluğunu kontrol etmektir. Metot: Seri dilüsyon metodu ile 4 tane Pseudomonas suju izole eidilmiştir. Bunların arasından Pseudomonas sp. 2 en iyi α-amilaz üretimini vermiştir. Bu suj, buğday kepeğinde SSF ile enzim üretiminde kullanılmıştır. Sonuçlar ve Tartışma: Optimum enzim üretimi 37 ºC and pH 7.5'da elde edilmiştir. Vitamin B 12 (50 mg) ve takiben piridoksin (50 mg) muamelelerinin enzim üretimini arttırdığı bulunmuştur. Sistein (30 mg), ıirozin (40 mg) ve alanin (30 mg) amino asitlerinin de enzim üretimini arttırdığı gözlenmiştir. Enzim, amonyum sülfat presipitasyonu ve DEAE-selüloz kolon kromatografisi ile kısmen saflaştırılmıştır. Saflaştırma verimi, ham enzimden 45.2 kez fazladır. SDS-PAGE kullanılarak enzimin molekül ağırlığı ~62 kDa bulunmuştur. Bu çalışma, Rizosfer'den elde edilen Pseudomonas sp.'dan ilk defa amilaz elde edilmesi ve kullanılmasını sunmaktadır. Kısmen saflaştırılmış enzim termostabil değildir, fakat aside dayanıklıdır ki bu enzimi endüstriyel uygulamalar için uygun hale getirmektedir. Anahtar Kelimeler: Pseudomonas sp. 2, α-amilaz, SSF, Vitaminler, Aminoasitler, Kısmi saflaştırma. Çıkar çatışması: Yazarlar hiçbir çıkar çatışması bulunmadığını beyan ederler.

Acid stable α-amylase from Pseudomonas balearica VITPS19—Production, purification and characterization

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“…Optimal temperature for amylase produced by Pseudomonas sp . 2 was approximately 30 °C [ 34 ]. But, Pseudomonas aeruginosa JCM5962 and Pseudomonas luteola amylases showed maximum activity at 40 °C and 60 °C respectively [ 32 , 35 ].…”

Purification and Characterization of Extracellular α-amylase from Aspergillus niger K8

“…The unincubated enzyme activity was taken as 100% for calculating the residual activities. Due et al (2008) mentioned that α-Amylase exhibited the maximum enzyme stability at 55 o C. However, Varalakshmi et al (2012) stated that when amylase enzyme was subjected to temperature stability studies, it was found that the enzyme was not thermostable. At 30ºC, 40ºC and 50ºC, the enzyme activity decreased with time.…”

“…pH 5.5 resulted in maximum enzyme activity. There was a 3.25 fold in enzyme activity after incubating the enzyme with a buffer at pH 5 (Varalakshmi et al, 2012). Relative activity (%)…”

Physicochemical and Microbial Screening of Palm Oil Mill Effluents for Amylase Production

“…Pathak and Narula (2013) studied the effect of pH on amylase production from soil mycotic flora and found out that Aspergillus flavus, Aspergillus niger and Rhizopus species were good amylase producers at different pH range. Varalakshmi et al (2012) reported that Pseudomonas species 2 produced α-amylase from rhizosphere soil under diverse conditions of temperature and pH. Farid and Shata (2011) reported that diverse strains of Aspergillus oryzae and Aspergillus awamori produce amylase.…”